Solid-phase immunoassays, based either on enzymes or radioactive isotopes as labels have found wide application in diagnostic microbiology due to their high specificity and sensitivity.
The specificity of an immunoassay is determined by the antibody or antigen which has been immobilized on the solid support. A major advantage of a solid phase assay is that on completion of the immune reaction, unwanted material is easily and rapidly separated from the antigen-antibody immune complex by a simple washing step. A wide variety of solid supports have found application for antibody or antigen immobilization and include polystyrene, polyvinyl chloride, nylon, titanous hydroxide, agarose beads and nitrocellulose.
The successful application of immunoassays to the detection of microorganisms in a sample is possible only if the particular organism of interest is present in sufficient numbers. This critical concentration is determined by the sensitivity of the immunoassay which can vary greatly depending on the affinity and avidity of a particular antibody for its antigen. It is for this reason that many immunoassays require culturing of the sample prior to performing the test.
This usually involves a pre-enrichment step to resuscitate injured microorganisms followed by a selective enrichment to increase the numbers of the microorganism of interest. Selecting culture conditions which favour the growth of a particular microorganism over its competitors has traditionally involved the use of either antibiotics, specific nutritional requirements or manipulation of the physical characteristics of the growth medium, e.g. temperature. These methods can take one to two days or several weeks depending on the organism and the extent of contamination with other microflora.
The use of immunosorbents for selecting microorganisms from a mixed population is known. The sorbent immobilizes a predetermined species of microorganism against which the antibodies are directed. Cells captured in this way can be incubated in a growth medium and then counted by such traditional techniques as plating and colony counting.
U.S. Pat. No. 4,592,994 describes a method for the determination or identification of microorganisms or unicellular organisms in a sample. The method involves exposing the sample to an absorbent having "a specific binding power" which may be provided by an antibody raised against the microorganism to be detected. Unbound sample is separated and the adsorbent with bound organisms is exposed to a nutrient medium to initiate metabolism. This nutrient medium undergoes physical or chemical changes as a result of this metabolism and these changes are observed in conjunction with calibration curves to determine presence and amount of the relevant microorganism. The assay involves indirect detection of original numbers of organisms through detection of metabolites in the medium. This may generate problems with regard to the specificity of the assay since different microorganisms may share metabolites.
U.S. Pat. No. 4,563,418 describes a method for the detection of a particular motile organism in a sample, for example, flagellate bacteria such as Salmonella species.
The method involves enriching the sample in an enrichment medium selective for the particular motile organism and filling a motility vessel with a non-selective medium containing a chemotactic attractant which serves to temporarily immobilize the organism of interest and its competitors in the medium for some time after inoculation. Antibodies specific to the flagella of the particular motile microorganisms are added through another opening in the motility vessel. The vessel is incubated under sufficient temperature and time conditions to permit the motile organisms to metabolize the chemotactic attractant, thereby reducing its concentration sufficiently to allow movement of the organisms present with the result that the organisms move through the medium and the particular motile organisms being assayed are immobilized by the antibodies. The quantity of antibody used is sufficient to produce a permanent immobilization band.
In Mohit et al ["A Simple Single-step Immunoimmobilization Method for the Detection of Salmonella in the Presence of Large Numbers of Other Bacteria" J. Med. Microbiol. 8 173 (1975)], a method of detecting Salmonella in a mixed population is described. This method employs a selective semi-solid medium, which promotes the migration of Salmonella, followed by immobilization using polyvalent H antisera.
La Roche et al in "Field Evaluation of the Membrane Filter-Disc Immobilization Technique in the Detection of Salmonella in Egg Products" describes a method for detecting Salmonella which involves using a membrane filter to concentrate Salmonella from a primary enrichment broth before selective migration in order to increase recovery.
Stannard reported in the annual report for Leatherhead RA for 1986, investigations into separation of Salmonella from other organisms, with a view to reducing the time required for detection of Salmonella in samples.
This method relied on antibody-coated magnetic particles. However, it was found that the apparent enrichment of Salmonella over other closely related organisms was in fact due to differential affinity of these organisms to the glassware used in the experiment. Attempts to use this differential affinity for glass to enrich Salmonella were unsuccessful due to the lack of specificity of the effect.
Thus it can be seen that the prior art methods for detecting microorganisms from mixed populations provide means for detecting low numbers where enrichment in selective medium is used. Immunoimmobilization has been used but has not been shown to be effective for detecting low numbers in the absence of such a selection on selective medium.
The current invention provides methods for detecting low numbers of a particular microrganism or microorganisms in a mixed population which overcome the need for pre-selection in selective media by using an immunoimmobilization technique followed by non-selective growth and immunoassay or by cleavage of the antibody-microorganism bond and growth of the microorganisms on non-selective media.
The methods can be especially usefully applied to detecting Salmonella and Listeria spp meeting the need for rapid, sensitive methods for their detection in mixed populations.